Naturally occurring regulatory T cells (Treg) are critical for maintaining a balance between beneficial and harmful autoreactive T cell responses. Treg cells (formerly called suppressor T cells) generally are responsible for controlling autoreactive T cell responses in a variety of immune responses. The existence of sub-populations of T cells that specialize in the suppression of immune responses was previously speculated in the early 1970s. Following these observations, “suppressor T cells” were found to be depressed in psoriasis and atopic dermatitis. It has been previously demonstrated, using allogeneic mixed lymphocyte reactivity (Allo-MLR) reactions, that T cells isolated from peripheral blood mononuclear cells (PBMC) of patients with psoriasis were less efficient at suppressing an MLR reaction than non-psoriatic suppressors. However, as with many early suppressor T cell studies, there was no further investigation of the role of suppressor T cells in psoriasis or dermatitis.
Recently, suppressor T cells have re-emerged as regulatory T cells (Treg) because a distinct phenotype was identified. Several recent reviews have described the re-emerging idea of T cells that regulate self-reactive T cells to maintain peripheral tolerance (non-autoimmune recognition of organ antigens). The identification of this class of Treg cell in human peripheral blood has also re-energized the field to concentrate on the potential mechanism(s) of action of this class of cell. Impaired capacity or removal of Treg cells allows excessive proliferative responses of pathogenic T cells.
Several subsets of Treg cells have been described and much progress has been made in understanding their ontogeny, function, and mechanisms of action. One such subset is the naturally occurring CD4(+)CD25(+) Treg that arise in the thymus and express the transcription factor Foxp3. However, Treg can also be induced in the periphery after immune activation. One such subset of Treg cells produce immunoregulatory cytokines, such as Interleukin-10 (IL-10) and are referred to as Tr1 cells. Regulatory T cells have also been classified as CD8+ Treg and TGF-β producing cells (Th3 cells), and exert their suppressive functions at least in part via the effects of these cytokines. The historical background and the spectrum of these T cell populations to which regulatory functions have been attributed, are well known in the art.
CD4+ cells that constitutively express the Interleukin-2 receptor (IL-2R) α-chain (CD25) have been identified as Treg cells in mice. This population was initially identified by its ability to inhibit the development of autoimmune gastritis that develops following neonatal thymectomy, and is capable of being enriched within the 10% of peripheral CD4+ cells that express CD25. CD4+CD25+ Treg cells also show a remarkable suppressive capacity both in vitro and in vivo. Transfer of these Treg cells reduces the pathology of experimentally induced autoimmune diseases such as gastritis, insulin-dependent diabetes mellitus, and colitis, whereas depletion of CD4+CD25+ Treg cells results in the development of systemic autoimmune diseases.
Treg cell function is a critical determinant of recrudescence in infections with intracellular microbes, in chronic inflammatory diseases, and in cancers. Thus, the natural presence of Treg cells in the immune system makes them a good target to manipulate and control pathologic as well as physiologic immune responses. However, strategic manipulation of Treg cells requires appropriate cell surface markers to distinguish them from non-regulatory T cells.
Treg cells are identifiable as CD4+ T cells that express relatively high levels of CD25. However, since activated T cells also generally express CD25, CD25 expression is normally not specific to Treg cells. It is known that expression of forkhead/winged-helix transcription factor FOXP3 differentiates Treg cells from activated T cells. FOXP3 is a member of a large family of transcription factors that drive numerous cellular responses and is critical for the development of murine CD4+CD25+ Treg cells and its high expression helps validate Treg cell purification. Recently, Foxp3 was identified as the gene responsible for an autoimmune condition in mice termed “scurfy”, where mutation results in lethality 15-20 days following birth, induced by multi-organ infiltration of over-proliferating CD4+ T cells. Foxp3 knockout mice (Foxp3−/−) exhibit lymphoproliferative disorders of the same magnitude as scurfy mice. Interestingly, adoptive transfer of wildtype lymphocytes protected the scurfy mice from developing disease. It was also observed, that the knockout Foxp3 mice do not possess a regulatory T cell (CD4+CD25+) population, in contrast to their wildtype counterparts, and, as with the scurfy animals, Foxp3−/− mice were protected by adoptive transfer of wildtype lymphocytes. The gene is highly conserved in humans and rodents, and mutations within FOXP3 result in a severe human autoimmune syndrome referred to as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome). However the protein product of the FOXP3 gene, scurfin, is intracellularly expressed, and thus has limited utility for manipulation of viable cells.
Currently, CD25 expression is considered to be the most suitable cell surface marker for regulatory T cells, representing 5-10% of CD4+ T cells in normal naive mice. It is unknown, however, what percentage of CD25+ cells are regulatory T cells within the human CD4+ subset, because CD25 is upregulated on activated for memory type T cells, as well. Without previous T cell receptor (TCR) stimulation, human CD25high+ cells express high levels of intracellular and low levels of cell surface CTLA-4 (CD152), and constitutively express CD122 (IL-2R beta chain), CD45RO, HLA-DR, CD62L and CD71 in the physiologic state that is commonly used to identify naturally occurring Treg cells. However, these cell surface receptors are also upregulated on activated non-regulatory T cells. While regulatory T cell function in mice has been well described, revealing the identity and function of their human counterparts has been controversial because of a lack of specific markers to accurately identify this subset. Therefore, there is a need for identification of alternative markers for CD25high+ Treg cells to facilitate the progress of research in naturally occurring human regulatory T cells. Recently, it was disclosed that regulatory T cells may be identified by consistent lack of expression of the IL-7R (CD127) which in combination may mark Treg cells more specifically than CD4CD25 staining. However, this selection process will not allow for the direct selection of Treg cells.
Murine CD4+CD25+ Treg cells are anergic (have a depressed response to antigens or are non-responsive to antigens) when stimulated in vitro with anti-CD3 mAbs, but proliferate upon addition of exogenous interleukin-2 (IL-2). After TCR-mediated stimulation, CD4+CD25+Treg cells suppress the activation and proliferation of other CD4 and CD8 T cells in antigen specific and antigen-nonspecific manners via a mechanism that requires cell—cell contact and, in most systems, is independent of production of immunosuppressive cytokines. Murine CD4+CD25+ Treg cells uniquely constitutively express cytoplasmic cytotoxic T lymphocyte-associated antigen (CTLA)-4, a receptor for the co-stimulatory ligands B7-1 and B7-2 and a negative regulator of T cell activation. Expression of this molecule may be required for these cells to suppress immune responses in vivo.
Leucine rich repeat containing (LRRC) proteins exhibit 20-29 leucine repeat motifs. These motifs are present in many proteins that participate in protein-protein interactions and exhibit various functions and are found in various cellular locations. LRRC 3D structural units consist of a beta strand (LxxLxLxxN/CxL conserved pattern) and an alpha helix. This results in a curved overall structure with parallel f3 sheets on the concave side, and helices on the convex side of the curve. This “finger-like” repeating structure's primary function is to provide a framework for protein-protein interaction. The “horseshoe” like shape of the LRRC facilitates these protein interactions. Several LRRC subfamilies have been identified, with the most recent consensus of seven distinct subgroups. LRRC 3D structures have been described and LRRC regions have been identified in numerous proteins including, ribonuclease inhibitors, proteoglycans, tyrosine kinase receptors and Toll like receptors. LRRC proteins are proposed to participate in biological activities, such as receptor hormone binding, cell adhesion, enzyme reactivity and cell trafficking. LRRC proteins have also been demonstrated to participate in neural development, apoptosis and regulation of gene expression. It is believed that in all of these interactions, LRRC domains facilitate protein-protein interaction.